Elsevier

Vaccine

Volume 25, Issue 49, 28 November 2007, Pages 8338-8345
Vaccine

Safety and efficacy in geese of a PER.C6-based inactivated West Nile virus vaccine

https://doi.org/10.1016/j.vaccine.2007.09.055Get rights and content

Abstract

Studies were performed with an inactivated vaccine against the mosquito-borne flavivirus, West Nile virus (WNV). The mammalian cell line, PER.C6®, was selected as the platform for WNV growth since both the neurovirulent strains NY99 and ISR98 that cause epidemics in humans and high mortality in geese, respectively, could be propagated to high titers (109 to 1010 TCID50/ml) on these cells. Based on the high DNA homology of the WNV envelope (E) protein and non-structural protein 5 (NS5), and identical neurovirulence in mice and geese, we concluded that NY99 and ISR98 viruses are closely related and therefore vaccine studies were performed with ISR98 as a model for NY99. A robust challenge model in domestic geese was set up resulting in 100% mortality within 7 days of intracranial challenge with 500 TCID50 WNV. Geese were used to assess the efficacy and safety of an inactivated WNV vaccine produced on PER.C6® cells. Efficacy studies demonstrated 91.4% (53/58) protection of geese compared to no protection (0/13) in geese receiving a sham vaccine. A follow-up study in 1800 geese showed that the vaccine was safe with a survival rate of 96.6% (95% lower CL 95.7%). Initial studies on the correlates of protection induced by the vaccine indicate an important role for antibodies since geese were protected when injected intra-cranial with a mixture of serum from vaccinated, non-challenged geese and WNV. In all, these results provide a scientific basis for the development of an inactivated WNV vaccine based on NY99 produced on PER.C6® cells for human and equine use.

Introduction

West Nile virus (WNV) was first identified in 1937 in the West Nile district of Uganda [1], and the first human outbreaks were reported in 1950 in Israel and 1974 in South Africa. These first outbreaks were associated only with mild flu-like symptoms with mortality rates close to zero [2], [3], [4], and as a result WNV was never considered as a serious threat to human populations. In 1999, a deadly variant of the WNV emerged in New York and spread rapidly into and across North America [5]. During this period, WNV has infected more than 120,000 individuals in the US leading to many hospitalizations. Since an effective human vaccine against WNV is not yet available, massive aerial spraying programs with pesticides aimed at eradicating the mosquito vectors were executed with limited effect. This aggressive emergence of WNV has prompted research laboratories to develop effective candidate vaccines for animal populations, notably horses, susceptible wild and domesticated avian species, and humans. The human disease caused by WNV (strain NY99) is characterized by fever, nausea and headache, and in many instances is further accompanied by diarrhea. In severe disease, WNV infects motor neurons in the brainstem resulting in the loss of neuron function. This leads to severe encephalitis and meningitis with mortality rates of 5–13%, rising to 15–30% in the geriatric population, children, and the immunocompromised [6], [7], [8], [9]. In horses, WNV causes a neurological disorder involving the spinal cord alone or the entire nervous system [10], [11]. Among the domesticated avian species affected by WNV, the goose is highly suscepible to natural infection. Clinical signs include ataxia, drooped wings and paresis. Recumbent geese are unable to stand and will die from secondary infection. In some young flocks up to 60% become affected and die [12]. Here, we show that WNV strains can be efficiently propagated on the mammalian PER.C6 cell line [13], and after inactivation and formulation with an adjuvant, the WNV vaccine demonstrates excellent safety in geese. Moreover, we show that vaccinated geese are fully protected against an otherwise lethal WNV challenge. Finally, we demonstrate here that protection of the geese correlates with antibody levels induced by the inactivated vaccine. These studies provide a scientific basis for the further development of an inactivated WNV vaccine based on strain NY99 which can then be tested in equines and in human clinical trials.

Section snippets

Virus strains and phylogenetic relationships

WNV strain ISR98 has been described previously [14] and was isolated in 1997 and 1998 from a dead goose in Israel [15]. Strains NY99 (snowy owl 385-99), AUS60 (MRM16 KUN), AUS91 (K6453 KUN), CYP68 (Q3574-5), and MAD78 (DakAnMg798) were provided by Dr. Robert Shope (University of Texas Medical Branch, Galveston, USA). To verify strain origin PCR primers were designed flanking the prM/E (nucleotides 549–1828) and NS5 (nucleotides 7681–10,395) genes as identified from GenBank entry AF196835

WNV replication in PER.C6® cells and estimation of goose and mouse LD50

To investigate whether WNV can replicate on mammalian PER.C6 cells we assessed the optimal inoculation dose of ISR98 on the cell line and showed that one WNV infectious particle per 104 PER.C6 cells causes 100% cell kill within 3 days (Fig. 1A). We subsequently determined the peak titers of six different WNV strains on PER.C6 cells. Although all tested strains could be propagated, the highest titers (mean log10 titer of 9.7 TCID50/ml) were attained for the neuro-invasive strains NY99 and ISR98 (

Discussion

The data obtained here demonstrate that the PER.C6 cell line provides an excellent platform for the production of an inactivated WNV vaccine. The major advantages of using PER.C6 cells include the excellent documentation of the origins of the cell line and its safety record which facilitates regulatory acceptance of products derived from this cell line. Also, it has been shown that this cell line can be scaled to grow in very large volumes, at high cell densities, and in diverse cell culture

Acknowledgements

The authors thank Dr. Robert Shope (University of Texas Medical Branch, Galveston, USA) for sending strains NY99, AUS60, AUS91, CYP68 (Q3574-5), and MAD78. The authors also thank Guiseppe Marzio and Marco Oerlemans (Crucell Holland BV) for technical support. This work was in part funded by SenterNovem grant IS04210.

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